Glatiramer acetate (Copaxone®) induces degenerate,
`Th2-polarized immune responses in patients
`with multiple sclerosis
`
`Petra W. Duda, Mascha C. Schmied, Sandra L. Cook, Jeffrey I. Krieger,
`and David A. Hafler
`
`Laboratory of Molecular Immunology, Center for Neurologic Diseases, Brigham and Women’s Hospital and
`Harvard Medical School, Boston, Massachusetts 02115, USA
`Petra W. Duda’s present address is: Neurology and Experimental Immunology, Department of Research,
`University Hospital Basel, 4031 Basel, Switzerland.
`
`Address correspondence to: David A. Hafler, Center for Neurologic Diseases, Harvard Institutes of Medicine,
`Room 780, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, USA.
`Phone: (617) 525-5330; Fax: (617) 525-5333; E-mail: hafler@cnd.bwh.harvard.edu.
`
`Petra W. Duda and Mascha C. Schmied contributed equally to this work.
`
`Received for publication November 23, 1999, and accepted in revised form February 15, 2000.
`
`We examined the effect of glatiramer acetate, a random copolymer of alanine, lysine, glutamic acid,
`and tyrosine, on antigen-specific T-cell responses in patients with multiple sclerosis (MS). Glatiramer
`acetate (Copaxone) functioned as a universal antigen, inducing proliferation, independent of any
`prior exposure to the polymer, in T-cell lines prepared from MS or healthy subjects. However, for most
`patients, daily injections of glatiramer acetate abolished this T-cell response and promoted the secre-
`tion of IL-5 and IL-13, which are characteristic of Th2 cells. The surviving glatiramer acetate–reac-
`tive T cells exhibited a greater degree of degeneracy as measured by cross-reactive responses to com-
`binatorial peptide libraries. Thus, it appears that, in some individuals, in vivo administration of
`glatiramer acetate induces highly cross-reactive T cells that secrete Th2 cytokines. To our knowledge,
`glatiramer acetate is the first agent that suppresses human autoimmune disease and alters immune
`function by engaging the T-cell receptor. This compound may be useful in a variety of autoimmune
`disorders in which immune deviation to a Th2 type of response is desirable.
`J. Clin. Invest. 105:967–976 (2000).
`
`Introduction
`Multiple sclerosis (MS) is an inflammatory disease of
`the central nervous system (CNS) white matter. The
`high frequency of activated, myelin-reactive T cells in
`the circulation and cerebrospinal fluid of patients
`with MS is consistent with the hypothesis that an ini-
`tiating event linked to an antecedent microbial infec-
`tion in a genetically susceptible host eventually leads
`to an autoimmune-mediated destruction of myelin
`followed by the surrounding axons (1). After the initi-
`ating event(s), the CNS itself may become a potential
`depot of antigen and MHC, with expression of critical
`second signals required for T-cell activation such as
`B7-1 and CD40 (2, 3) leading to epitope spreading (4).
`MS is thought to be a Th1-mediated disease based
`largely on pathological resemblance to a delayed-type
`hypersensitivity response in the CNS and from obser-
`vations made in the murine experimental autoim-
`mune encephelomyelitis (EAE) model. However, direct
`cloning of myelin-reactive T cells from the blood of
`patients with MS suggests that the majority of T cells
`can secrete both Th1- and Th2-type cytokines (5).
`A major goal in the treatment of autoimmune dis-
`eases has been the development of antigen-specific
`
`therapies that target autoreactive T cells. The discovery
`of epitope spreading in the EAE model (4, 6) and obser-
`vations of diverse T-cell receptor repertoires in response
`to self-antigens have theoretically made this approach
`less attractive. Instead, the concept of bystander sup-
`pression has emerged in which autoreactive Th2 or Th3
`T cells are generated that migrate to the inflamed tar-
`get organ where they are antigen specifically reactivat-
`ed, leading to the secretion of cytokines that downreg-
`ulate inflammation in the local milieu in an antigen
`nonspecific mechanism (7). Two approaches have
`emerged for inducing immune deviation of autoreac-
`tive T cells: mucosal administration of antigen, which
`induces Th2 T-cell responses to the antigen (7), and
`altered peptide ligands (APLs), which, by inducing a
`weaker strength of signal, lead to Th2 deviation of
`cytokine secretion (8–11). Both approaches have been
`used in clinical trials to treat patients with MS, but to
`date, without success.
`An alternative approach to the use of a single self-
`antigen that has been altered or given mucosally is the
`administration of peptide mixtures that contain many
`different antigen specificities. The use of random co-
`polymers that contain amino acids commonly used as
`
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`mechanism of action of GA involved the induction of
`regulatory T cells. Later, adoptive transfer of GA-spe-
`cific T cells was found to inhibit EAE (22). It was orig-
`inally thought that GA was structurally cross-reactive
`with MBP, although this has remained controversial.
`Recently, TCR antagonism has been suggested to
`occur in addition to competition for MHC binding
`(23). Stimulation of murine GA-reactive T-cell lines
`and clones with MBP was reported to induce the secre-
`tion of Th2 and Th3 cytokines to this cross-reactive
`antigen (24).
`Taken together, these data led to the hypothesis that
`GA acts as an APL in vivo, leading to alterations of
`responses to myelin antigens by cytokine deviation of
`myelin-specific T cells and bystander suppression
`mediated by GA-reactive T cells. Here, we directly test-
`ed this hypothesis by investigating changes in antigen-
`specific responses in patients with MS who were under-
`going treatment with daily subcutaneous injections of
`GA. T-cell reactivity to GA, the immunodominant MBP
`epitope 84–102 as a model myelin antigen, combinato-
`rial libraries derived from the MBP 84-102 sequence,
`and a completely random 13mer sequence was exam-
`ined in vitro before and during a year of treatment.
`Examination of proliferative responses to combinato-
`rial libraries was deemed potentially informative based
`on the observation that combinatorial peptide libraries
`are a powerful tool to examine the degree of T-cell
`receptor degeneracy. That is, the degree to which a T-
`cell clone proliferates to a random combinatorial pep-
`tide library where all of the 13 amino acids are random,
`representing a total of 1913independent peptides, to a
`first approximation provides information regarding
`the degree of degeneracy for that clone, i.e., the more
`peptides the clone can recognize, the more degenerate
`the T-cell receptor. Together, these experiments
`enabled us to examine whether daily subcutaneous
`injections of GA induced alterations of the T-cell
`immune response.
`
`Methods
`Patients. Patients with RR MS in early stages of the dis-
`ease, with MRI findings consistent with the diagnosis,
`and who decided with their physicians to initiate GA
`treatment participated in the trial. No clinical exami-
`
`Table 1
`Characteristics of the study patients
`
`MHC anchors and T-cell receptor (TCR) contact
`residues are possible “universal APLs.” Glatiramer
`acetate (GA) (Copaxone; Teva Marion Partners, Kansas
`City, Missouri, USA) (12) is a random sequence
`polypeptide of the 4 amino acids alanine (A), lysine (K),
`glutamate (E), and tyrosine (Y) at a molar ratio of
`A/K/E/Y of 4.5:3.6:1.5:1, respectively, and an average
`length of 40–100 amino acids. Directly labeled GA effi-
`ciently binds to different murine H-2 I-A molecules and
`to the human counterparts, MHC class II DR, but not
`to DQ or MHC class I, molecules in vitro (13). Bio-
`chemical studies revealed that GA also binds directly
`and with high affinity to purified HLA-DR1, -DR2, and
`-DR4 (14), suggesting that GA contains multiple epi-
`topes enabling it to bind promiscuously to MHC class
`II molecules, where it could potentially be recognized
`by CD4 T cells.
`A “universal antigen” containing multiple epitopes
`would be expected to induce proliferation in vitro, as
`measured by [3H]thymidine incorporation in naive T
`cells from the circulation, representing a high degree
`of cross-reactivity to other peptide antigens. In in vitro
`cultures of PBMCs from healthy humans, a strong
`dose-dependent proliferative response to GA has been
`reported (15). Similarly in our own studies, we found
`that GA elicits dose-dependent responses in all of
`more than 50 humans, including healthy subjects and
`patients with relapsing remitting (RR) and chronic
`progressive MS (P.W. Duda and D.A. Hafler, manu-
`script in preparation). The response to GA could be
`blocked by anti-DR antibodies and the restriction of
`GA-reactive CD4+ T cells to a particular HLA DR mol-
`ecule could be shown on a clonal level. The high pro-
`liferative and cytokine responses of naive PBMC CD4+
`T cells suggest a high frequency of circulating GA-reac-
`tive precursor T cells. Our own limiting dilution analy-
`sis suggests that the precursor frequency of GA-reac-
`tive T cells ranges from 1:5,000 to 1:100,000 PBMCs.
`Thus, GA appears to constitute a highly cross-reactive
`antigen preparation.
`In animal models of MS, prophylactic subcutaneous
`administration of GA has been shown to prevent EAE
`induced by injection of purified myelin basic protein
`(MBP) (12), proteolipid protein (PLP) (16), or myelin
`oligodendrocyte glycoprotein (MOG) (17). Of greater
`importance, in a phase III clinical trial
`subcutaneous administration of GA has
`been shown to decrease the rate of exac-
`erbations and to decrease the appear-
`ance of new lesions, based on magnetic
`resonance imaging (MRI), of patients
`with RR MS (18–20). This represents
`perhaps the first successful use of an
`agent that ameliorates autoimmune dis-
`ease by altering signals presumably
`through the TCR.
`The early observation that cyclophos-
`phamide abrogated the beneficial effect
`of GA on EAE (21), suggested that the
`
`Patient
`1
`2
`3
`4
`5
`6
`7
`
`Sex
`F
`M
`F
`F
`M
`F
`F
`
`DR
`
`3/13
`2/4
`2/4
`3/4
`1/11
`2/4
`2/8
`
`Age at
`onset of disease
`42
`48
`36
`35
`28
`45
`24
`
`Duration
`of disease (y)
`7
`10
`2
`1
`2
`1
`2
`
`EDSSA
`
`AttacksB
`
`1
`3
`1
`2
`1
`1
`0
`
`4
`0
`3
`1
`2
`2
`0
`
`968
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`The Journal of Clinical Investigation | April 2000 | Volume 105 | Number 7
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`AAssessed before initiation of treatment. BNumber of attacks in the 2 years before initiation of treatment.
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`Figure 1
`The proliferative response to GA is
`decreased on average after daily injec-
`tions of GA. The antigen-specific prolif-
`erative response of 20 or 30 primary T-
`cell lines induced with 40 m g/mL GA as
`described in Methods was measured by
`split-well assay for each patient at each
`time point. Before and at 6 months of
`treatment, all 7 patients could be tested;
`at 3 and 12 months, data for 5 patients
`were obtained. (a) Each panel represents
`data from an individual patient. Squares
`represent mean ± SEM proliferation in D
`cpm of the GA-specific response com-
`pared with the no-antigen control. Back-
`ground levels of the [3H]thymidine incor-
`poration for all patients of the no-antigen
`condition were 1,747 ± 111 before treat-
`ment, and 3,286 ± 175, 3,785 ± 262,
`and 3,509 ± 239 at 3, 6, and 12 months
`of treatment, respectively. The numbers
`in the figure indicate the SI over the no-
`antigen control for each time point. (b)
`The mean stimulation index SI ± SEM for
`all T-cell lines from all patients tested at
`each time point is shown.
`
`nation other than routine follow-up in the clinic was
`performed, and no other preselection criteria were
`applied. Informed consent was obtained before enroll-
`ment, and the study was performed in compliance with
`the rules of the ethical guidelines for human experi-
`ments of the Institutional Review Board of the
`Brigham and Women’s Hospital. Table 1 summarizes
`the patient characteristics.
`Antigens. GA (Copaxone; lots 123211 and 123243) was
`supplied by Teva Marion Partners. MBP 84-102 (DEN-
`PVVHFFKNIVTPRTPP) and MBP 93R (ENPVVHFFRNIVT-
`PR) peptides were synthesized by standard fmoc tech-
`nology and HPLC-purified to greater than 99%.
`Combinatorial peptide library X13 was a 13mer ran-
`domized at each position, and combinatorial peptide
`libraries with random amino acids inserted at position
`X of the MBP 85-99 peptide (ENPVVHFFKNIVTPR) were:
`90X (ENPVVXFFKNIVTPR), 91X (ENPVVHXFKNIVTPR),
`93X (ENPVVHFFXNIVTPR), 90X93R (ENPVVXFFRNIVT-
`PR), and 91X93R (ENPVVHXFRNIVTPR). All peptide
`libraries were obtained from Chiron Technologies
`(Raleigh, North Carolina, USA). Peptides were dissolved
`at 10 mg/mL in DMSO.
`Generation of antigen-specific T-cell lines. PBMCs were
`isolated from fresh drawn heparinized blood by
`Ficoll-Paque (Amersham Pharmacia Biotech, Uppsala,
`Sweden) gradient centrifugation according to manu-
`facturer’s protocol. Antigen-specific T-cell lines were
`generated by culturing 150,000 PBMCs per well in the
`presence of 40 m g/mL GA or MBP 84-102 peptide.
`Unless otherwise indicated, all cell cultures were done
`in 96-well U-bottom microtiter plates in 200 m L com-
`
`plete medium (RPMI 1640; BioWhittaker Inc., Walk-
`ersville, Maryland, USA) containing 2.5% heat-inacti-
`vated pooled human AB serum (PelFreeze, Brown
`Deer, Wisconsin, USA), sodium pyruvate, HEPES,
`nonessential amino acids, and glutamine) in a humid-
`ified 8% CO2 incubator at 37°C. For patient 5, whole
`human MBP was used instead of the MBP 84-102
`peptide throughout. On day 5 of culture, 120 m L of
`culture supernatants was removed and replaced by
`140 m L complete medium containing 10% phyto-
`hemagglutinin-free (PHA-free) T-stim (Collaborative
`Biomedical Laboratories, Bedford, Massachusetts,
`USA). On day 7, each GA-induced T-cell line was
`transferred into 1 mL of complete medium contain-
`ing 10% T-stim for further expansion. On day 12,
`split-well assays were performed to test for antigen-
`specific and cross-reactive proliferation and cytokine
`secretion. For patients 1, 2, 3, and 4, 40 T-cells lines to
`MBP 84-102 and 20 T-cell lines to GA were generated
`at each time point. For patients 5, 6, and 7, 30 T-cell
`lines were generated for each antigen.
`Cross-reactivity assays. Equal aliquots of primary T-cell
`lines were stimulated with irradiated (33 Gy) autolo-
`gous PBMCs that had been preincubated with antigen
`in 96-well U-bottom microtiter and ELISPOT plates at
`37°C for 1 hour. The conditions tested with primary
`GA-reactive lines were 20 m g/mL GA, 100 m g/mL X13,
`40 m g/mL MBP 84-102, 20 m g/mL 90X, 20 m g/mL 91X,
`20 m g/mL 93X, 20 m g/mL 93R, 20 m g/mL 90X93R, 20
`m g/mL 91X93R, and the no-antigen control. Primary
`MBP 84-102–induced lines were tested with 20 m g/mL
`MBP 84-102, 40 m g/mL GA, and no antigen.
`
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`Proliferation assay and cytokine measurement by ELISA.
`Equal aliquots of primary T-cell lines were stimulated
`with antigen-pulsed autologous PBMCs (100,000 per
`well). After 48 hours, 160 m L of supernatant was
`removed and frozen at –80°C for future cytokine analy-
`sis. The cells were pulsed with 1 m Ci/well of [3H]thymi-
`dine in 100 m L of complete medium. After a further 24
`hours, cells were harvested onto filter paper, and incor-
`poration of [3H]thymidine was measured in a scintilla-
`tion counter (Wallace, Gaithersburg, Maryland, USA).
`Supernatants were tested for cytokines in duplicate by
`performing standard sandwich ELISA using matched
`antibody pairs according to the manufacturer’s protocol
`(Endogen Inc., Woburn, Massachusetts, USA).
`ELISPOT assay. ELISPOT plates (Millipore Corp., Bed-
`ford, Massachusetts, USA) were coated with an optimal
`concentration of 100 m L of primary antibody diluted in
`0.1 mM NaHCO3 (pH 8.3) and incubated overnight at
`4°C. Antibody pairs were the same as those used in the
`sandwich ELISA assay described earlier here. Plates
`were washed 3 times with PBS and blocked with 1%
`BSA in HBSS at 37°C for 1 hour. Plates were again
`washed with PBS 3 times, and antigen-presenting cells
`were added together with antigen and placed in a 37°C,
`8% CO2 incubator for 60 minutes. Responder T cells
`were added, and plates were placed in a 37°C incubator
`for 24 hours. The plates were then washed 3 times with
`TP buffer (0.05% Tween in PBS) and incubated with
`100 m L of biotinylated secondary antibody in TP buffer
`overnight at 4°C. Plates were washed again 3 times
`with TP buffer and incubated at room temperature
`with 100 m L of a 1:1,000 dilution of streptavidin alka-
`line phosphatase conjugate Extravidin (Sigma Chemi-
`cal Co., St. Louis, Missouri, USA) for 2 hours. Plates
`
`were washed, and viewing of spots was carried out with
`100 m L BCIP/NBT substrate (Sigma Chemical Co.) pre-
`pared according to manufacturer’s instructions and by
`developing in dark for up to 20 minutes. The reaction
`was stopped by washing the plates with distilled water.
`Measurement of T-cell proliferation in primary in vitro cul-
`ture. PBMCs, isolated from heparinized blood as already
`described here, were incubated at 50,000 PBMCs per
`well in the presence of PHA or at 150,000 PBMCs per
`well in the presence of tetanus toxoid (Massachusetts
`Biological Laboratories, Jamaica Plain, Massachusetts,
`USA). On day 6, 160 m L of culture supernatant was
`removed from each well and replaced by 100 m L of com-
`plete medium containing 1 m Ci of [3H]thymidine. After
`further incubation at 37°C for 18 hours, cells were har-
`vested onto filter paper, and thymidine incorporation
`was measured by scintillation counting.
`HLA typing. Determination of the HLA DR and DQ
`phenotypes of each patient was determined by stan-
`dard PCR and hybridization methods.
`Statistical analysis. Statistical analysis was performed
`using the STATISTICA for Macintosh package (Stat-
`Soft, Tulsa, Oklahoma, USA) as indicated. Unless oth-
`erwise indicated, results are given as mean ± SEM.
`
`Results
`The in vitro proliferative response of PBMCs to GA decreases
`upon in vivo administration of GA. PBMCs were isolated
`from 7 patients with RR MS before and at various
`times after subcutaneous administration of GA. At
`each time point tested, primary and secondary in vitro
`proliferation and cytokine assays in the presence and
`absence of GA were performed. We found that before
`treatment, there was a significant proliferative
`
`Figure 2
`GA-specific secretion of cytokines is polarized toward a Th2 response after daily injections of GA. The GA-specific secretion of the cytokines
`IL-5 and IFN-g was measured in T-cell lines by 2 methods: ELISPOT (a) and ELISA (b) assays. Each symbol represents the difference of spots
`counted or D pg/mL measured in split-well assays between the GA (20 m g/mL) condition and the no-antigen control. The limits of detec-
`tion were 1 spot and 10 pg/mL, respectively. Numbers represent the percentage of T-cell lines in each quadrant with a minimum difference
`in spots of twice the SD of the negative controls for IL-5 and IFN-g , respectively.
`
`970
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`Figure 3
`IL-13 secretion is increased
`after daily injections of GA.
`Primary T-cell lines were set
`up in 10 identical wells each in
`the presence of no antigen or
`with 1.0, 10, and 100 m g/mL
`GA and cultured as described
`in Methods. Identical wells
`were pooled on day 11, and
`30,000 T cells each were res-
`timulated with no antigen or
`1.0, 10, and 100 m g/mL GA
`pulsed on 100,000 autolo-
`gous APCs. Cytokines were
`measured in supernatants by
`ELISA after 48 hours as
`described, and proliferation
`was measured by [3H]thymi-
`dine incorporation. Asterisks
`point at values that were
`above the upper limit of
`detection of the IL-13 assay of
`10,000 pg/mL.
`
`response as measured by [3H]thymidine incorporation
`to GA in all 7 patients, with an average stimulation
`index (SI) in vitro of 24.8 ± 1.1; the average D cpm was
`37,241 ± 1,766 cpm. Additionally, all of a total of 170
`independently derived T-cell lines stimulated in pri-
`mary in vitro culture with GA proliferated in response
`to the antigen (data not shown). After treatment with
`GA 20 mg subcutaneously daily for 3, 6 and 12
`months, the proliferative response as measured by SI
`and D
`cpm significantly decreased (Figure 1a) (P <
`0.001), although, as expected, individual patients var-
`ied in their response to GA (Figure 1b).
`In vitro–generated GA-reactive T-cell lines deviate toward a
`Th2-cytokine profile upon treatment with GA. Having
`demonstrated that the proliferative response to GA
`changed after in vivo subcutaneous administration of
`GA, we next examined whether the cytokine profile also
`changed. The cytokine response was measured for the
`prototypic Th1 and Th2 cytokines IFN-g and IL-5 by
`ELISPOT and sandwich ELISA in a total of 590 T-cell
`lines generated before and at various times after GA
`injection in all 7 patients. As shown in Figure 2, a and
`b, compared with the values detected before treatment,
`the average IFN-g secretion to GA measured by either
`ELISA or ELISPOT was significantly decreased (P <
`0.001 by Tukey’s honest statistical difference test) after
`treatment for 3, 6, and 12 months, except for the meas-
`urement by ELISPOT at 3 months, which did not reach
`significance, and the ELISA measurement at 6 months,
`which only reached a significance level of P < 0.01. The
`GA-dependent IFN-g
`secretion as determined by
`
`ELISPOT as D
`spots between the cells tested with anti-
`gen and the no-antigen control was 104 ± 10 before
`treatment, 132 ± 18 at 3 months, 28 ± 4 at 6 months,
`and 18 ± 3 at 12 months. IFN-g secretion measured by
`sandwich ELISA in D pg/mL was 1,405 ± 150 before
`treatment, 222 ± 38 at 3 months, 797 ± 185 at 6
`months, and 5.9 ± 46 at 12 months. When patients
`were analyzed individually, 5 of the 7 patients had sta-
`tistically significant decreases in IFN-g secretion (P <
`0.001) by ELISPOT or sandwich ELISA (data not
`shown). The levels of IFN-g secretion were correlated
`with the decreased proliferative capacity in these
`patients (r2 of > 0.8 for ELISA values in all patients test-
`ed, measurement by ELISPOT correlated less well with
`r2> 0.5 in 4 patients). This is in accordance with previ-
`ous observations that the proliferative and IFN-g
`responses are correlated (25).
`GA-specific IL-5 secretion was, on average, not sta-
`tistically significantly altered during treatment with
`GA. By ELISPOT assay, the average IL-5 secretion in D
`spots was 10 ± 3 before, 10 ± 2 at 3 months, 11 ± 2 at 6
`months, and 6 ± 1 at 12 months after the initiation of
`treatment. When measured by sandwich ELISA as D
`pg/mL, IL-5 secretion was 1,484 ± 266 before treat-
`ment; 1,940 ± 554 at 3 months; 1,738 ± 343 at 6
`months; and 1,146 ± 303 at 12 months after the initi-
`ation of treatment. One patient had a sustained sta-
`tistically significant decrease in IL-5 (P < 0.001), and 1
`had a sustained statistically significant increase (P <
`0.001) in IL-5 secretion as measured by ELISPOT dur-
`ing treatment.
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`Figure 4
`Cross-reactivity of GA-reactive T-cell lines is increased after daily injections of GA. Percentages of the GA-induced T-cell lines cross-react-
`ing to each APL tested at each time point are shown for the 7 patients encoded by gray scale. Proliferative IFN-g and IL-5 responses were
`examined for all T-cell lines and are represented separately in the top, middle, and lower third. A minimum SI of 2 and a difference of
`2 SD over the background was required for classification as a cross-reactive T-cell line.
`
`To examine further the cytokine pattern of PBMCs
`from patients before and after treatment with GA, T-cell
`lines from all subjects were grouped into Th0, Th1, and
`Th2 subsets, based on their cytokine profile. T-cell lines
`were considered positive for a cytokine when the differ-
`ence of the GA condition was increased at least 2-fold
`over the SD of the no-antigen controls. Thus, values
`over background considered positive were 19 spots for
`IFN-g and 10 spots for the IL-5 ELISPOT assay, and 138
`pg/mL for IFN-g and 485 pg/mL for the IL-5 ELISA
`assays. When measured by ELISPOT before treatment,
`46% of all T cells evaluated were characterized as Th1
`(Figure 2a). During the course of treatment, there was
`an increased proportion at 3 months of Th0-type T-cell
`lines and at 6 and 12 months of Th2-type T-cell lines. In
`parallel with the decreased proliferative response, an
`increasing proportion of T-cell lines that did not secrete
`either IFN-g or IL-5 in response to GA was seen with
`treatment. Measurement by ELISA appears to be slight-
`ly more sensitive in this assay in which 39% of T-cell
`lines secreted Th0 cytokines and 41% secreted Th1
`cytokines before treatment (Figure 2b). A similar shift
`
`toward a Th2 response as with the ELISPOT assay with
`a decrease of Th0 and a simultaneous increase of Th2-
`type T-cell lines at 3, 6, and 12 months of treatment was
`seen. When classifications of T-cell lines into Th0, Th1,
`and Th2 were performed under less-stringent condi-
`tions (minimum difference of 20 and 10 ELISPOTS for
`IFN-g and IL-5, respectively, or 200 pg/mL in ELISA
`assays), a cytokine shift toward Th0/Th2 could also be
`confirmed (data not shown).
`As the magnitude of the IL-5 response was uniform-
`ly low, especially as measured by ELISPOT, it was
`important to reconfirm the apparent Th2 deviation
`with GA treatment by a means other than measuring
`IL-5. Toward this end, 3 new patients with RR MS were
`recruited to the study, and serial measurements of IFN-
`g and IL-13 secretion were made before and after the
`initiation of daily subcutaneous GA injections. IL-13
`was chosen as the candidate Th2 cytokine because
`there are no IL-13 receptors on T cells to consume the
`secreted cytokine. Primary in vitro T-cell lines generat-
`ed in the presence of no antigen and of 1.0, 10, and 100
`m g/mL GA were examined at 30,000 PBMCs per well
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`each with the 4 antigen concentrations. Thus, equal
`numbers of cells were tested in the secondary stimula-
`tion, whereas in the previous assay with equal aliquots
`of primary cell lines, the number of cells tested
`decreased, on average, with treatment owing to lower
`expansion in the primary cultures. A marked increase
`in IL-13 secretion in 2 of 3 patients after 3 months of
`therapy was observed. IFN-g secretion and the prolifer-
`ative response were stable or decreased when compared
`with the pretreatment values (Figure 3).
`Cross-reactivity of GA-reactive T-cell lines is increased upon
`treatment with GA. Cross-reactivity of GA-induced T-cell
`lines to combinatorial peptide libraries derived from
`the immunodominant MBP 84-102 peptide and a com-
`pletely randomized 13mer library were performed to
`determine degeneracy of GA-specific T cells (Figure 4).
`Before treatment with GA, there was minimal cross-
`reactivity in either the proliferative or the cytokine
`responses. There was only 1 instance when antigen
`cross-reactivity was observed, in the proliferative
`response to the 93R90X combinatorial peptide library
`in patient 7. In striking contrast, 6 of the 7 patients
`demonstrated an increased number of cross-reactive T-
`
`cell lines after therapy to the combinatorial peptide
`libraries. However, no dominantly cross-reactive APL
`emerged from this analysis, consistent with the degen-
`erate immune responses we observed.
`In vitro T-cell reactivity to the immunodominant epitope
`MBP 84-102 is not significantly altered during GA therapy.
`In contrast to the results from primary in vitro cul-
`tures with GA, which is well approximated by a nor-
`mal distribution of responses owing to the high pre-
`cursor frequency of responsive T cells, the reactivity
`of primary T-cell lines to MBP 84-102 was low and as
`such not normally distributed. Therefore, nonpara-
`metric statistics were used for analysis. No signifi-
`cant change over time of treatment was seen for the
`MBP 84-102–specific proliferative responses (Figure
`5a) by Mann-Whitney U tests. Further analysis
`included percentage of MBP 84-102–specific T-cell
`lines, which were not significantly changed over the
`course of treatment.
`Analysis of cytokine secretion in response to MBP 84-
`102 by Mann-Whitney U test also did not reveal any sig-
`nificant differences during the course of treatment with
`one exception: IL-5 measured by ELISPOT at 12
`
`Figure 5
`(a). The proliferative response to MBP 84-102 is not altered after daily injections of GA. A total of 900 T-cell lines were generated in response to MBP
`84-102. The proliferative response in D cpm is given for each of 30 or 40 lines tested at each time point (circles, D cpm on left axes). On the right axes,
`the percentage of positive lines determined by a minimum 2.5-fold increase over background and a minimum difference of 1,500 cpm is shown (line).
`The mean background was 4,662 ± 138. (b). No changes occur in the cytokine response to MBP 84-102 after daily injections of GA. Lines having a
`minimum difference to the negative control of 2 SD, i.e., 70 spots for IFN-g and 19 spots for IL-5, were considered positive for the respective cytokine.
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`months tested significantly decreased (P < 0.01) com-
`pared with pretreatment values. When the cytokines
`were analyzed for each patient individually, no signifi-
`cant change in the frequency of Th0-, Th1-, and Th2-
`cytokine–secreting lines was seen (Figure 5b), regardless
`of the stringency of the criteria used for classification.
`No cross-reactivity was seen between MBP 84-
`102–induced T cell lines and GA in vitro when judged
`by these criteria.
`GA treatment does not change tetanus toxoid–specific in vitro
`T-cell responses. To examine the effects of GA treatment
`on the primary in vitro T-cell response to an unrelated
`recall antigen, PBMCs from 6 patients with RR MS who
`were undergoing GA therapy were analyzed with
`tetanus toxoid. A dose-dependent proliferative response
`to 0.3 m g/mL, 3 m g/mL, and 30 m g/mL of tetanus toxoid
`as well as to 0.1 m g/mL and 1 m g/mL of the PHA control
`was observed at all time points. Compared with the pre-
`treatment values, no significant differences in the
`tetanus toxoid–specific proliferative responses were seen
`during treatment with GA as determined by the Stu-
`dent’s t test (data not shown). Thus, as with MBP 84-
`102, subcutaneous treatment with GA did not alter
`immune responses to a common recall antigen.
`
`Discussion
`We examined the effect of daily subcutaneous injec-
`tions of GA on antigen-specific T-cell responses in
`patients with MS. GA appears to function as a univer-
`sal antigen, inducing primary in vitro proliferation of
`naive T-cell populations both in patients with MS and
`in normal healthy controls. Daily subcutaneous injec-
`tions of GA caused a striking loss of in vitro respon-
`siveness to the GA that was accompanied by immune
`deviation to a more Th2 type of response. The surviv-
`ing GA-reactive T cells exhibited a greater degree of
`degeneracy as measured by cross-reactive responses to
`combinatorial peptide libraries. GA is, to our knowl-
`edge, the first agent effective in the treatment of an
`autoimmune disease that appears to alter immune
`function by engagement of the T-cell receptor and may
`be useful in a variety of autoimmune disorders in
`which immune deviation to a Th2 type of response
`may be desirable.
`Perhaps the most striking observation in these inves-
`tigations was the ability of GA to induce the prolifera-
`tion of non-GA–primed T-cell populations, which then
`decreased with treatment. This was not a nonspecific
`mitogenic response, as MHC DR-restricted T-cell
`clones could be generated with in vitro antigen culture
`(P.W. Duda, and D.A. Hafler, manuscript in prepara-
`tion). GA has been shown to directly bind different DR
`molecules without antigen processing, allowing many
`potential interactions with T-cell receptors (14).
`Although it had been thought that the recognition by
`the TCR of an MHC/peptide complex was highly spe-
`cific, it has recently become clear that there can be
`extensive degeneracy in TCR recognition of antigen in
`the trimolecular complex (26, 27). Moreover, we recent-
`
`ly demonstrated at a functional level that a TCR that
`may appear to be highly specific for 1 peptide/MHC
`complex may become significantly more degenerate in
`its recognition of antigen with subtle changes of amino
`acid side chains, particularl